Research Article
Intervertebral Disk Mediated Postoperative Epidural Fibrosis: Experimental Model and Methods of Prevention
Sergey N. Larionov1,2*, Vladimir A. Sorokovikov1,
2Department of Traumatology, Orthopedics and Neurosurgery, Irkutsk State Medical Academy of Continuous
Education, Russia
*Corresponding author: Sergey N. Larionov, Department of Neurosurgery, Irkutsk Scientific Center of Surgery and Traumatology, Bortzov Revolutzii 1, Irkutsk 3, 664003; Russia
Published: 25 May, 2017
Cite this article as: Larionov SN, Sorokovikov VA,
Erdyneyev KC, Lepekhova SA,
Goldberg OA, Rudakova AV.
Intervertebral Disk Mediated
Postoperative Epidural Fibrosis:
Experimental Model and Methods of
Prevention. Clin Surg. 2017; 2: 1484.
Abstract
The etiology and pathogenesis creates a lot of discussion, and selection of methods of treatment and prevention continues. In this study we used 36 male Wistar rats, each weighing 250 ± 30 g; LIV laminectomy with dura mater exposition was done in 12 rats, and then, 0.3 cc of elements of suspension of autologous intervertebral disk were implicated on dura mater. In 12 animals laminectomy was followed by application of a 5 × 6 mm sheet of Reperen™ on the exposed dura™. Histological assessment of the lumbar spine of rats observed that the elements of autologous intervertebral disk play the role of inflammation trigger, which cause postoperative scar and epidural fibrosis. In another part of the experimental study we revealed that Reperen™ to prevent excessive epidural fibrosis. So, the experimental model and methods of prevention postoperative epidural fibrosis can be widely used in experimental medicine for research of epidural scar-adhesion pathogenesis and for searching the ways of treatment and prevention of the disease in human. Keywords: Experimental model; Postoperative epidural fibrosis; Laminectomy; Intervertebral disk
Introduction
Surgical treatment of herniated lumbar disk is one of the most common spine operations. The procedure does not always result in pain-relief, and epidural scar formation is one of the reasons why this treatment fails. Epidural fibrosis has been described in 24–38% of patients with failed back surgery syndrome [1-3]. Re-operations, aimed at adhesiolysis and scar resection, are difficult, have higher risk of complications and often are ineffective [4,5]. Although surgical intervention is important for the pathogenesis of postsurgical forms of epidural fibrosis, some aspects of this disorder are difficult to explain basing only on wound healing process [6,7]. Literature data analysis shows that there are different inflammatory substances involved in formation of scar adhesions after spinal surgery, and various degrees of peridural fibrosis are detected [2,8,9]. Therefore, the investigations and ongoing experimental studies of pathogenesis of postoperative epidural scar formation are issues of current interest in modern neurosurgery. They are necessary for further search for adequate model of intervertebral disk mediating postoperative epidural fibrosis and developing new methods of treatment and prevention of epidural fibrosis after intervertebral disk hernia surgery [10-13]. The purpose of our study was to work out a new experimental model of epidural fibrosis and its prevention. Model is necessary for the development of adequate methods of postoperative epidural scar prevention.
Materials and Methods
The study was approved by the Ethical Committee of Irkutsk Scientific Center of Surgery and
Traumatology (N 5, 05.11.2011). In this study we used 36 male Wistar rats, each weighing 250±30 g,
and the animals were allocated into three groups. Prior the operation all rats endured intramuscular
cefazolin sodium injection (20 mg/kg). The animals were anesthetized with intraperitoneally
administrated ketamine hydrochloride (2 mg/kg) (Ketalar; Pfizer Inc., USA) and were fixed on the
operation table in the prone position. Following sterile isolation, 3 cm midline surgical incisions
were performed between the first and the fifth lumbar vertebrae. The paravertebral muscles were
dissected, exposing LIV laminae. For performing laminectomy we used a high-speed electrical
drill, and in all cases dura mater was exposed. LIV laminectomy with dura mater exposition was
done in 12 rats (group 1), and then, 0.3 cc of elements of suspension of autologous intervertebral disk were implicated on dura mater (10). In group 2 of animals
(n=12) laminectomy was followed by application of a 5 × 6 mm
sheet of Reperen™ on the exposed dura, and then, 0.3 cc of elements
of suspension of autologous intervertebral disk were implicated on
the sheet of Reperen™. As an autologous intervertebral disk we used
intervertebral disk from amputated tail. The elements of autologous
intervertebral disc were suspended and applied on dura mater. In all
animals incisions were closed with 3/0 vicryl. Then the rats were left
for free food and water consumption. 6 rats in groups 1 and 2 were
sacrificed 30 days after the surgery and the rest – after 60 days. In the
control group, LIV laminectomy was performed in 12 rats. Six rats
of the control group were sacrificed in 30 days after surgery and the
rest – in 60 days.
The animals were sacrificed with intraperitoneally administered
thiopental sodium solution (10 mg/kg). After narcotization, all
blood was removed from their abdominal aorta. Then we exposed
paravertebral region and resected vertebral column including
paraspinal muscle (between the ThX and LVI levels) in an en bloc
fashion (8). The tissue samples were fixated with 10% formaldehyde
solution and decalcified, 5 micron thick sections were stained with
Hematoxylin-Eosin (H&E). The preparations were examined under a
light microscope with magnification × 40, 100, 200 and 400. Epidural
fibrosis was examined according to the following scheme designed by
Y He et al. [10]: Grade 0 – dura mater is free of scar tissue; Grade 1 –
only thin fibrous bands are observed between the scar tissue and dura
mater; Grade 2 – continuous adherence is observed in < two-thirds of
the laminectomy defect; and Grade 3 – scar tissue adherence is large,
affecting > two-thirds of the laminectomy defect, or the adherence
extended to the nerve roots (radix spinalis retraction) 10. Fibroblast
cell density was calculated in each field at × 40 magnification: Grade
1 – fewer than 100 fibroblasts in each field; Grade 2 – 100–150
fibroblasts in each field; Grade 3 – more than 150 fibroblasts in each
field.
Statistical analysis
We performed data analysis with the help of SPSS for Windows,
version 11.5 [SPSS Inc., Chicago, IL, USA]. The Shapiro-Wilk test was
used to determine normal distribution of continuous variables. The
Bartlett test was used to evaluate the homogeneity of the variances.
Continuous and ordinal variables were shown as medians (minmax).
Nonparametric Kruskal-Wallis test determined the statistical
significance of the epidural fibrosis extension*, inflammation, radix
spinalis retraction, dural adhesion†, and fibroblast cell density among
groups. To compare the differences in the median values among the
groups we used the Mann-Whitney U-test. The likelihood ratio test
was applied to determine whether the differences in nominal data
were statistically significant. When the p values from the likelihood
ratio test data were statistically significant, we employed Wilcoxon’s
exact test to determine which group differed from which of the other
groups. A p value less than 0.05 was considered statistically significant.
Figure 1
Figure 1
Grade 1 fibrosis as observed in the control group on the 30th day.
Less EF and fibroblast cell density and only thin fibrous bands between DM
and scar tissue are observed. Hematoxylin and Eosin, original magnification
× 40..
Figure 2
Figure 2
Grade 3 fibrosis as observed in the experimental group on the 30th
day. Dense EF, dural adhesion, and RS and DM retraction are seen in the
laminectomized area. Hematoxylin and Eosin, original magnification × 40.
Figure 3
Figure 3
Grade 2 fibrosis as observed in the Reperen™-treated group on
the 30th day. Hematoxylin and Eosin, original magnification × 40.
Table 1
Results
Histological assessment of the lumbar spine of rats under light
microscopy revealed various changes in the surgical field (Table 1).
Grade 1 of epidural fibrosis, marked only in one rat in group 1,
and was characterized by the presence of only thin fibrous bands
between scar tissue and dura mater (Figure 1).
It was established, that rough scar-adhesion changes prevailed
in group 1. Pathological changes corresponding to Grade 2 and 3 of
epidural fibrosis were revealed in 11 animals. Moreover, in 7 animals
with elements of the autologous intervertebral disk as an epidural
fibrosis trigger, adhesions filled more than 2/3 of laminectomy space
or spread to nerve roots (Figure 2). It was revealed that nerve roots
were adhered to dura mater in the spinal canal space and deformation
of the dural sac with rough adhesive processes and obliterated epidural
space were marked. Also the spinal canal defect was filled with lots of newly formed fibrous tissue, along the sides bounded by fragments
of yellow ligament. All these facts pointed to rapid development and
preservation of epidural fibrosis.
In the Reperen™-treated group of animals epidural scars were less
extensive than in the group 1. Pathological changes corresponding to
Grade 2 and 3 of epidural fibrosis were revealed in 5 animals. Grade
1 of epidural fibrosis, marked in 58.3% of animals in group 2, was
characterized by the existence of only thin fibrous bands between scar
tissue and the sheet of Reperen™ (Figure 3).
In the control group, Grade 1 dural adhesion was present in 9 rats
(75%), and Grade 2 was found in the rest 3 rats (25%). In group 1,
Grade 1 dural adhesions were observed in 1 rat (8.3%), Grade 2 – in 3
rats (25.0%), and Grade 3 – in 8 rats (66.7%). In the Reperen™-treated
group of rats, Grade 1 dural adhesions were observed in 7 rats (58.7%),
Grade 2 – in 3 rats (25.0%), and Grade 3 – in 2 rats (16.7%). In the
control group, radix spinalis retraction was marked in 1 rat (12.5%).
Whereas, in group 1 dural adhesion with radix spinalis retraction
were found in 7 rats (58.3%), but in the Reperen™-treated group
they were detected in 2 animals (16.7%). Fibroblast cell density and
epidural fibrosis were lower in group 2 than in group 1 (p=0.003, and
p=0.005, respectively); these differences were statistically significant.
There was significant concordance of all parameters between
control and group 1 observations (k coefficient 0.684, 0.712, 0.702,
and 0.502 for epidural fibrosis, dural adhesion, fibroblast cell density
and radix spinalis retraction, respectively). All of these values were
statistically significant (p< 0.001).
By the 60th day of the experiment scar formations in animals
of group 1 progressed both in epidural space and in dura mater.
Inflammation processes were of multiform character. Whereas in
the control and group 2 these changes were less expressed, which
suggested reduction of inflammation.
Cellular reaction and fibroblast proliferation decreased in all
groups and were less noticeable in group 1 in 60 days. Hyalinized
fibrous tissue, characterized by trabecular structure and osteoblastic
activity, filled the laminectomy defect. Between the animals of
the Reperen™-treated group and of group 1 we found important
differences regarding the fibroblast cell density and epidural fibrosis
(p=0.003 and p=0.035, respectively). Moreover, inflammation, dural
adhesion, and fibroblast cell density were observed significantly less
frequently in the Reperen™-treated group than in group 1 (p=0.042,
p=0.005, and p=0.004, respectively). These results showed statistically
significant difference between the parameters of fibroblast cell density
in the experimental and control groups (p< 0.05).
Discussion
It is known that scar tissue is always formed as a physiological
reaction to any surgical intervention in response to the surgical
trauma. However, the intensity and duration of this process may be
different and depends on many factors. At present, the reasons of
the epidural fibrosis cause a lot of discussion. La Rocca et al. [12].
stated that post-operative hematoma in epidural space that replaces
epidural adipose tissue and eventually leads to aseptic inflammation,
causes intensive scar formation and epidural fibrosis. Furthermore,
migration of fibroblasts from affected paraspinal muscles results
in enhanced collagen synthesis [13]. The ratio of cells and fibrous
structures changes and friable connective tissue transforms into
dense scar-adhesions. According to some researchers, patrimonial
factor associated with hyperergic reaction of fibroblasts as a response
to surgical trauma plays an important role as well [14-16]. In
addition, it is known that tissue of degenerated nucleus pulposus
can maintain a state of chronic inflammation in spinal canal and
nerve roots, membranes of spinal cord and epidural adipose tissue,
and it causes reactive changes therein, which leads to development
of scar-adhesions [14,17]. Intervertebral disk tissue is avascular; it is
formed separately from the immune system and possesses antigenic
properties. The destruction of intervertebral cartilage triggers the
cascade mechanism of cellular immunity, which leads to formation
of anti-disk antibodies. Antigen-antibody complexes stimulate the
production of proinflammatory substances (cytokines, prostaglandin
E) and proteolytic enzymes (proteases, collagenases), and that induces
progressive degeneration of the intervertebral disk and adhesions
with other structures of the spinal canal [4,6].
In modern science, researchers preserve high interest to this
subject due to the fact that development of epidural fibrosis is one of
the reasons of compression and fixation of neurovascular structures
and circular stenosis of spinal canal, leading respectively to pain
occurrence and neurological symptoms in patients undergoing spinal
surgery. The fact that epidural fibrosis is one of the reasons of the
failed back surgery syndrome is well known [2,5]. Thus, according to
various authors, postoperative epidural adhesions are responsible for
up to 25% of reoperations [6,8].
The mechanisms of development of postoperative epidural
adhesions are still unclear. There is a question what the reasons are
that some patients after spinal surgery have severe epidural fibrosis
with appropriate symptoms while in other cases the fibrosis is
minimal despite the same conditions. Neurosurgical investigations
of this issue are quite rare, so clinical and experimental experience
is minor [14-18], and further research in this area is required. Basing
on histological results we confirmed our model of experiment. On
the 30th day of evaluation there were significant histological evidences
of post-operative epidural adhesions in experimental animals, which
included the obliteration of epidural space, the presence of adhesions
in the dura and nerve roots, the restructuring of the yellow ligament,
bone sclerosis, excessive appearance of fibrous tissue around the
autologous intervertebral disk tissue that applied on the dura mater.
In another part of the experimental study we used Reperen™
to prevent excessive epidural fibrosis. The present study noted
statistically significant differences (p< 0.05) between the Reperen™-
treated group and group of animals with experimental epidural
fibrosis in relation to both components – fibroblast cell density and
epidural fibrosis.
As a potential sheet for human use, Reperen™ has a favorable profile;
it has demonstrated no significant toxicity (in the form of foreign
body reaction) in experimental subjects. Similarly, inflammation did
not develop after implantation of Gore-Tex, Adcon-L, or vicryl.
Conclusion
We can resume that the proposed method of postoperative epidural fibrosis modeling can be widely used in experimental medicine for research of epidural scar-adhesion pathogenesis and for searching the ways of treatment and prevention of the disease in human. Moreover, Reperen™ has a positive effect on dural and neural structures in the vertebral canal after lumbar disk spine surgery. So, this material may be used as one of the methods to prevent excessive scar hypertrophy after intervertebral disk surgery.
References
- Andersson GB. Epidemiological features of chronic low-back pain. Lancet. 1999;354(9178):581-5.
- Erdyneev KC, Sorokovikov VA, Larionov SN. The postoperative scar-adhesive epiduritis (review). Bull. VSNC SO RAMN. 2011;77:243-46.
- Rönnberg K, Lind B, Zoega B, Gadeholt-Göthlin G, Halldin K, Gellerstedt M, et al. Peridural scar and its relation to clinical outcome: a randomised study on surgically treated lumbar disc herniation patients. Eur Spine J. 2008;17(12):1714-20.
- Rodrigues FF, Dozza DC, de Oliveira CR, de Castro RG. Failed back surgery syndrome: casuistic and etiology. Arq Neuropsiquiatr. 2006;64(3B):757-61.
- Seelig W, Nidecker A. [Pain following operations of the lumbar spine. The "failed back surgery syndrome"]. Z Orthop Ihre Grenzgeb. 1989;127(3):346-53.
- Fritsch EW, Heisel J, Rupp S. The failed back surgery syndrome: reasons, intraoperative findings, and long-term results: a report of 182 operative treatments. Spine (Phila Pa 1976). 1996;21(5):626-33.
- Su WR, Lee JS, Chen HH, Wang LC, Huang YH, Jung YC, et al. Neurophysiological and Histopathological Evaluation of Low-Dose Radiation on the Cauda Equina and Postlaminotomy Fibrosis. An Experimental Study in the Rat. Spine. 2009;34(5):463-9.
- Fransen P. [Postoperative epidural fibrosis after lumbar disc surgery: fact or fiction?]. Rev Med Suisse. 2010;6(238):468-71.
- Larionov SN, Sorokovikov VA, Erdyneyev KC, Lepekhova SA, Goldberg OA. Experimental Model of Intervertebral Disk Mediated Postoperative Epidural Fibrosis. Ann Neurosci. 2016;23(2):76-80.
- He Y, Revel M, Loty B. A quantitative model of post-laminectomy scar formation. Effects of a nonsteroidal anti-inflammatory drug. Spine (Phila Pa 1976). 1995;20(5):557-63.
- Isık S, Taşkapılıoğlu MÖ, Atalay FO, Dogan S. Effects of cross-linked high-molecular-weight hyaluronic acid on epidural fibrosis: experimental study. J Neurosurg Spine. 2015;22(1):94-100.
- LaRocca H, Macnab I. The laminectomy membrane. Studies in its evolution, characteristics, effects and prophylaxis in dogs. J Bone Joint Surg Br. 1974;56(3):545-50.
- Topsakal C, Akpolat N, Erol FS, Mehmet Faik Ozveren, Ismail Akdemir, Metin Kaplan, et al. Seprafilm superior to Gore-Tex in the prevention of peridural fibrosis. J Neurosurg. 2004;101(2):295-302.
- Austin JW, Kang CE, Baumann MD, Di Diodato L. The effects of intrathecal injection of a hyaluronan-based hydrogel?on inflammation, scarring and neurobehavioural outcomes in a rat model of severe spinal cord injury associated with arachnoiditis. Biomaterials. 2012; 33:4555-64.
- Chen JM, Lee SH, Tsai TT, Niu CC, Chen LH, Chen WJ. Anti-adhesive effect of hyaluronate in a rabbit laminectomy model. Biomed J. 2014;37(4):218-24.
- Preul MC, Campbell PK, Garlick DS, Spetzler RF. Application of a new hydrogel dural sealant that reduces epidural adhesion formation: evaluation in a large animal laminectomy model. J Neurosurg Spine. 2010;12:381-90.
- Cemil B, Tun K, Kaptanoglu E, Kaymaz F, Cevirgen B, Comert A, et al. Use of pimecrolimus to prevent epidural fibrosis in a postlaminectomy rat model. J Neurosurg Spine. 2009;11(6):758-63.
- Yildiz KH, Gezen F, Is M, Dosoglu M. Mitomycin C, 5-fluorouracil, and cyclosporin A prevent epidural fibrosis in an experimental laminectomy model. Eur Spine J. 2007;16:1525-30.